Brazilian Journal of Pharmaceutical Sciences vol. 51, n. 2, apr./jun., 2015 Article http://dx.doi.org/10.1590/S1984-82502015000200005

Differential pulse voltammetric determination of diclofenac in pharmaceutical preparations and human serum

Bilal Yilmaz*, Selcuk Kaban, Bilge Kagan Akcay, Ulvihan Ciltas

Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, Erzurum, Turkey

This article describes a differential pulse voltammetric (DPV) method for the determination of diclofenac in pharmaceutical preparations and human serum. The proposed method was based on electro-oxidation

of diclofenac at platinum electrode in 0.1 M TBAClO4/acetonitrile solution. The well-defined two oxidation peaks were observed at 0.87 and 1.27 V, respectively. Calibration curves that obtained by using current values measured for second peak were linear over the concentration range of 1.5-17.5 μg mL-1 and 2-20 μg mL-1 in supporting electrolyte and serum, respectively. Precision and accuracy were also checked in all media. Intra- and inter-day precision values for diclofenac were less than 3.87, and accuracy (relative error) was better than 4.12%. The method developed in this study is accurate, precise and can be easily applied to Diclomec, Dicloflam and Voltaren tablets as pharmaceutical preparation. In addition, the proposed technique was successfully applied to spiked human serum samples. No electro- active interferences from the endogenous substances were found in human serum.

Uniterms: Diclofenac/determination. Differential pulse /quantitative analysis. Pharmaceutical formulations/analysis. Human serum/analysis

Este artigo descreve um método de voltametria de pulso diferencial (VPD) para a determinação de diclofenaco em preparações farmacêuticas e em soro humano. O método proposto foi baseado em

eletroxidação de diclofenaco no eléctrodo de platina em solução 0,1 M TBAClO4/acetonitrila. Dois picos de oxidação bem definidos foram observados em 0,87 e 1,27 V, respectivamente. As curvas de calibração obtidas utilizando-se valores de corrente medidos por segundo pico foram lineares no intervalo de concentração de 1,5-17,5 μg mL-1 e 2-20 μg mL-1 em eletrólito suporte e soro, respectivamente. Precisão e exatidão também foram verificadas em todos os meios. Valores de precisão intra- e inter-dia para o diclofenaco foram inferiores a 3.87 e a precisão (erro relativo) foi melhor do que 4,12%. O método desenvolvido neste estudo é exato, preciso e pode ser facilmente aplicado a Diclomec, Dicloflam e comprimidos Voltaren, como preparação farmacêutica. Além disso, a técnica proposta foi aplicada com sucesso em amostras de soro humano. Não se observaram interferências das substâncias endógenas no soro humano.

Unitermos: Diclofenaco/determinação. Voltametria de pulso diferencial/análise quantitativa. Formulações farmacêuticas/análise. Soro humano/análise.

INTRODUCTION It is widely used in clinical medicine for the treatment of inflammatory conditions such as rheumatoid arthritis, Diclofenac (Figure 1), [o-(2,6-dichloroanilino) osteoarthritis and ankylosing spondylitis (Gostick et al., phenyl] acetate), is a non-steroidal anti-inflammatory 1990; Crowley et al., 1990). The efficacy of diclofenac drug (NSAID) with strong anti-pyretic, analgesic and anti- equals that of many newer and established NSAIDs. As an inflammatory properties (Iliiescu, Baia, Miclaus, 2004). analgesic, it has a fast onset and a long duration of action. Compared to other NSAIDs, diclofenac is well tolerated and rarely produces gastrointestinal ulcerations or other ∗Correspondence: B. Yilmaz. Department of Analytical Chemistry. Fac- serious side effects. Thus, diclofenac can be considered as ulty of Pharmacy. Ataturk University. 25240 - Erzurum, Turkey. E-mail: [email protected] one of few non-steroidal anti-inflammatory drugs of first 286 B. Yilmaz, S. Kaban, B. K. Akcay, U. Ciltas

In recent years, applications of carbon nanotube modified carbon paste electrodes have provided considerable improvements in the electrochemical behaviour of biologically important compounds (Antiochia et al., 2004; Norouzi et al., 2007). Metal nanowires such as dysprosium showed behaviour like carbon nanotubes. A carbon- paste electrode containing 10% (w/w) of dysprosium nanowire/carbon paste electrode, in comparison with carbon-paste electrodes without nanowire, showed a very effective catalytic activity in the electrochemical oxidation of diclofenac. The reported methods were influenced by interference of endogenous substances and FIGURE 1 - Chemical structure of sodium diclofenac. potential loss of drugs in the re-extraction procedure and involving lengthy, tedious and time-consuming plasma choice used in the treatment of acute and chronic, painful sample preparation and extraction processes requiring a and inflammatory conditions (Roskar, Kmetec, 2003). sophisticated and expensive instrumentation. To date, several methods for the determination The development of a new method capable of of diclofenac have been reported. These include determining the drug amount in pharmaceutical dosage potentiometry (Shamsipur, Jalali, Ershad, 2005; Santini, forms is important. Electro-analytical techniques have Pezza, Pezza, 2006; Hassan et al., 2005), capillary zone been used for the determination of a wide range of electrophoresis (Jin, Zhang, 2000), high performance drug compounds with the advantages that there are, liquid chromatography (HPLC) (González, Yuln, Volonté, in most, instances no need for derivatization and that 1999; Kasperek, 2008; Arcelloni et al., 2001), high- these techniques are less sensitive to matrix effects than performance liquid chromatography-mass spectrometry other analytical techniques. Additionally, application (HPLC-MS) (Abdel-Hamid, Novotny, Hamza, 1990), of includes the determination of spectrophotometry (Botello, Perez-Caballero, 1995), electrode mechanism. Redox properties of drugs can spectrofluorometry (Arancibia, Boldrini, Escandar, 2000; give insights into their metabolic fate or their in vivo Damiani et al., 1999; Carreira et al., 1995), thin layer redox processes or pharmacological activity (Kissenger, chromatography (Sun, Fabre, 1994), gas chromatography Heineman, 1996; Kauffmann, Vire, 1993; Ozkan, Uslu, (Siou, Pommier, Godbillon, 1991; Yilmaz, 2010), Senturk, 2004). Despite the analytical importance of the polarographic analysis (Xu, Chen, Song, 2004), electrochemical behaviour and oxidation mechanism spectrophotometry (De Souza, Tubino, 2005; Agatonovic- of diclofenac, no report has been published on the Kustrin et al., 1997; Matin, Farajzadeh, Joyuban, 2005; voltammetric study of the electrochemical oxidation Sastry, Prasad-Tipirneni, Suryanarayana, 1989; Sena et of diclofenac in non-aqueous media. Diclofenac is not al., 2004) and electrochemistry methods (Blanco-Lopez et soluble in water. It can be dissolved in water containing al., 2003; Goyal, Chatterjee, Agrawal, 2010; Manea et al., 30% methanol. Methanol has a narrow potential range. 2010). In all electrochemical methods, modified electrodes Therefore, it was dissolved in acetonitrile which had wide were used due to the gradual passivation or contamination potential range. of unmodified solid working electrodes occurred during It is well known that the experimental and the diclofenac oxidation. The development of modified instrumental parameters directly affect the electrochemical electrodes is certainly one of the most extensive areas process and voltammetric response of drugs. Consequently, of research in analytical chemistry. However, it should it would be interesting to investigate the oxidation process be emphasized that electrode modification is frequently of diclofenac in aprotic media. Therefore, the goal of considered tedious and laborious process that can limit this work was the development of new DPV method for the use of these types of electrodes in routine analysis. the direct determination of diclofenac in pharmaceutical Carbon-paste electrodes, due to their ease of construction, preparations and spiked human serum samples without renewability and compatibility with various types of any time-consuming extraction or evaporation steps modifiers, have been widely used as a suitable matrix prior to drug assay. This paper describes fully validated, for the preparation of modified electrodes. Further, they simple, rapid, selective and sensitive procedures for show rather low background current compared to solid the determination of diclofenac employing DPV at the graphite or noble metal electrodes (Valentini et al., 2003). platinum disc electrode. Besides, the proposed method Differential pulse voltammetric determination of diclofenac in pharmaceutical preparations and human serum 287 might be an alternative to the chromatographic methods For quality control (QC) samples containing concentration in therapeutic drug monitoring. 4, 8 and 16 µg mL-1 of diclofenac, the stock solution was

diluted with 0.1 M TBAClO4/acetonitrile. MATERIAL AND METHODS Procedure for pharmaceutical preparations Chemicals and reagents A total 10 tablets of diclofenac (Diclomec, Dicloflam Diclofenac was obtained from Sigma (St. Louis, MO, and Voltaren) were accurately weighed and powdered. USA). Acetonitrile (Fluka for HPLC analysis) was purified An amount of this powder corresponding to one tablet by drying with calcium hydride, followed by distillation diclofenac content was weighed and accurately transferred from phosphorus pentoxide. Tetrabutylammonium into 100 mL calibrated flask and 50 mL of 0.1 M TBAClO4/ perchlorate (TBAClO4) were purchased from Fluka acetonitrile was added and then the flask was sonicated to and used as received without further purification. 10 min at room temperature. The flask was filled to volume

Diclomec, Dicloflam and Voltaren tablets were obtained with 0.1 M TBAClO4/acetonitrile. The resulting solutions from pharmacy (Erzurum, Turkey). Human serum in both cases were filtered through Whatman filter paper was obtained from Yakutiye blood bank, Erzurum, no 42 and suitably diluted to get final concentration within Turkey. the limits of linearity for the respective proposed method. The drug content of diclofenac tablets was calculated from Electrochemical instrumentation the current potential curve.

Electrochemical experiments were performed on Recovery studies a Gamry Potentiostat Interface 1000 controlled with software PHE 200 and PV 220. All measurements were To study the accuracy and reproducibility of the carried out in a single-compartment electrochemical cell proposed technique, recovery experiments were carried with a standard three-electrode arrangement. A platinum out using the standard addition method. In order to know disk with an area of 0.72 cm2 and a platinum wire were used whether the excipients show any interference with the as the working and the counter electrodes, respectively. analysis, known amounts of pure diclofenac were added The was successively polished with to the pre-analysed tablet formulation and the mixtures 1.0, 0.3 and 0.05 µm alumina slurries (Buehler) on were analysed by the proposed method. After five repeated microcloth pads (Buehler). After each polishing, the experiments, the recovery results were calculated using the electrode was washed with water and sonicated for 10 calibration equation. min in acetonitrile. Then, it was immersed into a hot piranha solution (3:1, H2SO4, 30% H2O2) for 10 min, and Analysis of spiked serum samples rinsed copiously with water. All potentials were reported versus Ag/AgCl/KCl (3.0 M) reference electrode (BAS Drug-free human blood, obtained from healthy Model MF-2078) at room temperature. The electrolyte volunteers (after obtaining their written consent), was solutions were degassed with purified nitrogen for 10 centrifuged (5000 rpm) for 30 min at room temperature min before each experiment and bubbled with nitrogen and separated serum samples were stored frozen until during the experiment. Operating conditions for DPV were assay. An aliquot volume of serum sample was fortified pulse amplitude 50 mV, pulse width 50 ms and scan rate with diclofenac dissolved in acetonitrile to achieve final 20 mV s-1. concentration of 100 µg mL-1. Acetonitrile removes serum proteins more effectively, as the addition of 1.0 mL volume Preparation of the standard and quality control of serum is sufficient to remove the proteins. After vortexing solutions for 30 s, the mixture was then centrifuged for 5 min at 5000 rpm for getting rid of serum protein residues and the The stock standard solution of diclofenac was supernatant was taken carefully. Appropriate volumes of prepared in 0.1 M TBAClO4/acetonitrile to a concentration this supernatant were transferred into the volumetric flask. of 100 µg mL-1 and stored at 4 °C. Working standard The concentration of diclofenac was varied in the range of solutions were prepared from the stock solution. A 2-20 µg mL-1 in human serum samples. Quantifications were calibration graph was constructed in the range of 1.5, 2.5, performed by means of calibration curve method from the 5, 7.5, 10, 12.5, 15 and 17.5 µg mL-1 for diclofenac (n=6). related calibration equation. 288 B. Yilmaz, S. Kaban, B. K. Akcay, U. Ciltas

RESULTS AND DISCUSSION

Voltammetric behaviour of diclofenac

The electrochemical behaviour of diclofenac was investigated at the Pt disc electrode in anhydrous acetonitrile solution containing 0.1 M TBAClO4 as the supporting electrolyte by using (CV). Figure 2 shows a typical cyclic voltammogram of 20 μg mL-1 diclofenac recorded under these conditions for the scan rate of 0.2 V s-1. In the anodic sweep, two oxidation peaks are seen at about the potentials of 0.87 and 1.27 V, respectively.

FIGURE 3 - Linear sweep voltammograms for the oxidation of -1 20 μg mL diclofenac in acetonitrile containing 0.1 M TBAClO4 as a function of scan rate.

considered. These results suggest that the redox species are diffusing freely from solution and not precipitating onto the electrode surface. The reason for this behaviour may be due to the solubility of the intermediate species in acetonitrile or poor adherence of products on the electrode surface. The oxidation peak potential (E) for the peaks shifts toward more positive values with increasing scan rate. The FIGURE 2 - Cyclic voltammogram for the oxidation of relationship between the peak potential and scan rate is -1 20 μg mL diclofenac in acetonitrile containing 0.1 M TBAClO4 described by the following equation, at Pt disk electrode, scan rate: 0.2 V s-1. 0’ 1/2 -1 E = E + RT/[(1 – )naF][0.78 + ln(D ks ) – In order to gain a deeper insight into the voltammetric 0.5 lnRT/[(1 – )naF]] + RT/[(1 – )naF]/2 lnv waves, the effect of scan rate on the anodic peak currents (I) and peak potentials (E) was studied in the range of and from the variation of peak potential with scan rate -1 0.01-1 V s of the potential scan rates in acetonitrile αna can be determined, where α is the transfer coefficient -1 solution containing 20 μg mL concentration of diclofenac and na is the number of electrons transferred in the rate (Figure 3). determining step. According to this equation, the plots of The representative linear sweep voltammograms the peak potentials versus ln ν for the oxidation peak show -1 obtained at Pt electrode for 20 μg mL diclofenac as a linear relationship. The slope indicates the value of αna is function of the scan rate are presented in Figure 4. Scan rate 0.38 for peak. On the basis of above results, na is 1 then dependency experiments show that the peak currents vary the value of α is calculated as 0.38, which is reasonable linearly with the scan rate (ν) (Figures 4a,b), which points for most irreversible electrode processes. Based on out the adsorption-controlled process. However, the plots of above discussions, the oxidation process of diclofenac is logarithm of peak currents versus logarithm of scan rates for controlled by the diffusion step and one electron and one 20 μg mL-1 concentration of diclofenac display straight lines proton are involved in the reaction. with 0.497 slope (Figure 4c), which are close to theoretical value of 0.5 expected for an ideal diffusion-controlled Analytical applications and validation of the electrode process (Laviron, Roullier, Degrand, 1980). proposed method Log I-log ν curve is more eligible for this aim, therefore, a diffusional process for peak should be In order to develop a voltammetric procedure for the Differential pulse voltammetric determination of diclofenac in pharmaceutical preparations and human serum 289

FIGURE 4 - A, B, C: Dependence of peak current on the scan rate (20 μg mL-1). determination of drug, we selected the DPV technique, since the peaks were sharper and better defined at lower concentration of diclofenac than those obtained by cyclic and with a lower background current, resulting in improved resolution. DPV is an effective and rapid electro-analytical technique with well- established advantages, including good discrimination against background currents and low detection limits (Wang, 1996; Kissenger,Heineman, 1996). Calibration graphs from the standard solution of diclofenac according to the procedures described above were constructed by using DPV. A linear relation in the concentration range between 1.5-17.5 μg mL-1 was found, indicating that the response was diffusion controlled in this range (Figure 5). Above this concentration (17.5 μg mL-1), a loss of linearity was probable due to the adsorption of diclofenac on the electrode surface. The characteristics of the calibration plots are summarized in Table I. The precision of the DPV method was determined FIGURE 5 - DPV voltammograms obtained for determination by repeatability (intra-day) and intermediate precision in supporting electrolyte (blank, 1.5, 2.5, 5, 7.5, 10, 12.5, 15 (inter-day). Repeatability was evaluated by analysing QC and 17.5 µg mL-1). samples six times per day, at three different concentrations which were QC samples. The intermediate precision where s is the standard deviation of the peak currents was evaluated by analysing the same samples once daily (three runs) and m is the slope of the calibration curve. for two days. The RSD of the predicted concentrations The LOD and LOQ values were also shown in Table I. from the regression equation was taken as precision. Repeating five experiments on 15 μg mL-1 diclofenac for The accuracy of this analytic method was assessed as the DPV technique tested the repeatability and reproducibility percentage relative error. For all concentrations studied, of peak potential and peak currents. The results were intra- and inter-day relative standard deviation values shown also in Table I. Repetition of the sample analysis were ≤3.87% and for all concentrations of diclofenac the after 72 h period did not show any significant change in relative errors were ≤4.12%. the results of the analyses. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated on the peak current Determination of diclofenac in tablets using the following equation: On the basis of above results, DPV method was LOD = 3 s/m; LOQ = 10 s/m applied for the direct determination of diclofenac in 290 B. Yilmaz, S. Kaban, B. K. Akcay, U. Ciltas

TABLE I - Regression data of the calibration lines for the quantitative determination of diclofenac

DPV Parameters Supporting electrolyte Serum Measured potential (V) 1.34 1.31 Linearity (μg mL-1) 1.5-17.5 2-20 Slope 6.7632 5.3249 Intercept 27.26 32.67 R 0.999 0.998 Sa 9.48 12.42 Sb 0.765 1.624 LOD (μg mL-1) 0.30 0.60 LOQ (μg mL-1) 0.90 1.80 Precision (RSD%) 2.96 3.87 Accuracy (% relative error) 3.46 4.12 Repeatability of peak current (RSD%)a 1.38 2.67 Repeatability of peak potential (RSD%) 1.94 2.46 Reproducibility of peak current (RSD%) 2.96 3.24 Reproducibility of peak potential (RSD%) 0.69 1.24 RSD: Relative standard deviation, aAverage of six replicate determinations, Sa: Standard deviation of intercept of regression line, Sb: Standard deviation of regression line slope, R: Correlation coefficient, LOD: Limit of detection, LOQ: Limit of quantification pharmaceutical preparations, using the related calibration There is no official method in any pharmacopoeias straight lines without any sample extraction or filtration (e.g. USP, BP or EP) or literature method related to and after adequate dilutions. The results show that the pharmaceutical dosage forms of diclofenac. To prove the proposed method was successfully applied for the assay of absence of interferences by excipients, recovery studies diclofenac in its pharmaceutical dosage forms (Table II). were carried out. The results demonstrate the validity of The accuracy of the method was determined by its the proposed method for the determination of diclofenac in recovery during spiked experiments. Recovery studies tablets. These results reveal that both methods had adequate were carried out after the addition of known amounts precision and accuracy and consequently can be applied to of the pure drug to various pre-analysed formulation of the determination of diclofenac in pharmaceuticals without diclofenac. According to the results, excipients presented any interference from the excipients. in tablet do not interfere with the analysis (Table II). Birajdar, Meyyanathan and Suresh (2010) developed

TABLE II - Recovery of the diclofenac in pharmaceutical preparations

Diclomec Dicloflam Voltaren Labelled claim (mg) 100 50 75 Amount found (mg)a 99.4 50.2 75.4 RSD% 2.43 1.84 2.73 Bias% -0.60 0.40 0.53 Added (μg mL-1) 10 10 10 Found (μg mL-1) 9.94 10.04 10.09 Recovery% 99.4 100.4 100.9 RSD% of recovery 2.52 1.92 2.32 a Each value is the mean of five experiments Differential pulse voltammetric determination of diclofenac in pharmaceutical preparations and human serum 291 a reverse phase HPLC method for the simultaneous tried as the serum precipitating agents. The best results determination of rabeprazole and diclofenac in tablet. The were obtained with acetonitrile. Hence, acetonitrile was method was based on HPLC separation of both drugs in used for the following studies. The measurements of reverse phase mode using Phenomenox C18 column with diclofenac in serum samples were performed as described Waters HPLC system by using mobile phase composition in the analysis of spiked serum samples section. For the of acetonitrile and 50 mM ammonium acetate buffer applicability of the proposed method to the human serum, (pH 3.6) (60:40 v/v) at flow rate 1mL min-1. Detection the calibration equation was obtained in spiked human wavelength used at 254 nm. Linearity was obtained in the serum. Calibration equation parameters and validation concentration range of 1.0-3.2 µg mL-1 for rabeprazole and data are shown in Table I. Obtained recovery results of 6.0-16.0 µg mL-1 diclofenac. spike human serum samples are given in Table III. Sastry, Prasad-Tipirneni and Suryanarayana (1989) described a spectrophotometric method for TABLE III - Recovery of diclofenac in human serum the determination of diclofenac sodium in bulk samples and pharmaceutical preparations with Added Found % Recovery % RSDb p-N,N‑dimethylphenylenediamine as solvent and (μg mL-1) (Mean ± SDa) maximum absorbance at 670 nm. The reaction is sensitive 2 1.86 ± 0.051 93.0 2.74 -1 enough to permit the determination of 2.0-24 µg mL . 5 4.83 ± 0.219 96.6 4.53 Agrawal Shivramchandra (1991) described two 7.5 7.45 ± 0.499 99.3 6.69 methods for the determination of diclofenac. In the first method diclofenac reduces iron(III) to iron(II) having 10 9.82 ± 0.785 98.2 7.99 a maximum absorbance at 520 nm. The reaction obeys 12.5 12.32 ± 1.08 98.6 8.77 Beer’s law for the concentrations of 10-80 µg mL-1. In 15 14.82 ± 1.29 98.8 8.70 the second method, diclofenac is treated with methylene 17.5 18.04 ± 1.67 103.7 9.25 blue in the presence of phosphate buffer (pH 6.8) and the 20 20.2 ± 1.65 101.0 8.17 complex is extracted with chloroform. The complex has a SDa: Standard deviation of six replicate determinations, maximum absorbance at 640 nm and linearity was in the RSD: Relative standard deviation, bAverage of six replicate -1 range 5-40 µg mL . determinations Thongchai et al. (2006) developed a high performance thin layer chromatographic method for the Using the proposed DPV technique, no sample determination of diclofenac sodium in pharmaceutical pre-treatment was required other than precipitation and formulations. The drug was extracted from the sample then dilution steps. The recovery results of diclofenac (Table various aliquots of this solution were spotted automatically III) in serum samples were calculated from the related by means of Camag Linomat IV on a silica gel 60 F254 linear regression equations, which are given in Table aluminium plate, using a mixture of toluene: ethyl acetate: I. Good recoveries of diclofenac were achieved from glacial acetic acid (60:40:1, v/v/v) as mobile phase. The this type of matrix. Analysis of serum samples by DPV spot areas were quantified by densitometry at 282 nm. involved only protein precipitation and centrifugation, Linear calibration curve was obtained over the range no time-consuming extraction and evaporation steps 5‑80 µg mL-1. are required. In this study, the serum proteins and For DPV measurements, calibration curve was endogenous substances in serum samples are precipitated linear over the concentration range of 1.5-17.5 μg mL-1 for by the addition of acetonitrile, which is centrifuged at diclofenac, which is as good as or superior to that reported 5000 rpm, and the supernatant was taken and diluted with in other papers (Birajdar, Meyyanathan, Suresh, 2010; the supporting electrolyte and directly analysed. Typical Sastry, Prasad-Tipirneni, Suryanarayana, 1989; Agrawal DPV curve of diclofenac examined in serum samples are et al., 1991; Thongchai et al., 2006). shown in Figure 6. As can be seen in Figure 6, no oxidation compounds Determination of diclofenac in human serum and no extra noise peaks present in biological material samples peak occurred in the potential range where the analytical peak appeared. The optimized procedure was successfully applied Stability of serum samples kept in refrigerator for the determination of diclofenac in protein-free spiked (+4 °C) was tested by making five consecutive analyses of human serum samples. Acetonitrile and methanol were the sample over a period of approximately 5 h. There were 292 B. Yilmaz, S. Kaban, B. K. Akcay, U. Ciltas

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